System ASC
amino acid transporter-2 (ASCT2) was previously demonstrated to be essential for human
hepatoma cell growth and survival, as its silencing via inducible
antisense RNA expression results in complete apoptosis within 48 h by a mechanism that transcends its role in
amino acid delivery. To gain mechanistic insights into the reliance of cancerous liver cells on ASCT2, the aim of this study was to determine the early consequences of its silencing on the growth and survival signaling that presage apoptosis. Induced antisense ASCT2
RNA in SK-Hep1 cells led to >90% suppression of ASCT2
mRNA by 6 h and inhibition of
mammalian target-of-rapamycin (mTOR)/raptor (mTOR complex-1;
mTORC1) signaling by 8 h, as manifested by diminished p70
ribosomal protein S6 kinase-1 and
eukaryotic initiation factor-4E (
eIF4E) binding protein-1 phosphorylation, while
protein synthesis rates declined by nearly 50% despite no measurable decreases in the
cap binding protein eIF4G or cellular
ribosomal protein content. Depressed
mTORC1 signaling occurred before detectable reduction in ASCT2 activity but coincided with a 30% decline in total cellular ASCT2
protein. By 12 h after ASCT2 silencing, further decrements were observed in
protein synthesis rates and ASCT2
protein and activity, each by approximately 50%, while signaling from mTOR/rictor (mTOR complex-2;
mTORC2) was stimulated as indexed by enhanced phosphorylation of the Akt/PKB
kinase on serine-473 and of its proapoptotic substrate Bad on serine-136. These results suggest that ASCT2 silencing inhibits
mTORC1 signaling to the translational machinery followed by an mTORC2-initiated survival response, establishing a link between
amino acid transporter expression and mTOR function.